Glycerol‐induced folding of unstructured disulfide‐deficient lysozyme into a native‐like conformation

2SS[6‐127,64‐80] variant of lysozyme which has two disulfide bridges, Cys6‐Cys127 and Cys64‐Cys80, and lacks the other two disulfide bridges, Cys30‐Cys115 and Cys76‐Cys94, was quite unstructured in water, but a part of the polypeptide chain was gradually frozen into a native‐like conformation with increasing glycerol concentration. It was monitored from the protection factors of amide hydrogens against H/D exchange. In solution containing various concentrations of glycerol, H/D exchange reactions were carried out at pH* 3.0 and 4°C. Then, ¹H‐¹⁵N‐HSQC spectra of partially deuterated protein were measured in a quenching buffer for H/D exchange (95% DMSO/5% D₂O mixture at pH* 5.5 adjusted with dichloroacetate). In a solution of 10% glycerol, the protection factors were nearly equal to 10 at most of residues. With increasing glycerol concentration, some selected regions were further protected, and their protection factors reached about a 1000 in 30% glycerol solution. The highly protected residues were included in A‐, B‐, and C‐helices and β3‐strand, and especially centered on Ile 55 and Leu 56. In 2SS[6‐127,64‐80], long‐range interactions were recovered due to the preferential hydration by glycerol in the hydrophobic box of the α‐domain. Glycerol‐induced recovering of the native‐like structure is discussed from the viewpoint of molten globules growing with the protein folding. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 665–675, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Evidence for an initiation site for hen lysozyme folding from the reduced form using its dissected peptide fragments.

We prepared two dissected fragments of hen lysozyme and examined whether or not these two fragments associated to form a native-like structure. One (Fragment I) is the peptide fragment Asn59-homoserine-105 containing Cys64-Cys80 and Cys76-Cys94. The other (Fragment II) is the peptide fragment Lys1-homoserine-58 connected by two disulfide bridges, Cys6-Cys127 and Cys30-Cys115, to the peptide fragment Asn106-Leu129. It was found that the Fragment I immobilized in the cuvette formed an equimolar complex with Fragment II (K(d) = 3.3x10(-4) M at pH 8 and 25 degrees C) by means of surface plasmon resonance. Moreover, from analyses by circular dichroism spectroscopy and ion-exchange chromatography of the mixture of Fragments I and II at pH 8 under non-reducing conditions, it was suggested that these fragments associated to give the native-like structure. However, the mutant Fragment I in which Cys64-Cys80 and Cys76-Cys94 are lacking owing to the mutation of Cys to Ala, or the mutant fragment in which Trp62 is mutated to Gly, did not form the native-like species with Fragment II, because the mutant Fragment I derived from mutant lysozymes had no local conformation due to mutations. Considering our previous results where the preferential oxidation of two inside disulfide bonds, Cys64-Cys80 and Cys76-Cys94, occurred in the refolding of the fully reduced Fragment I, we suggest that the peptide region corresponding to Fragment I is an initiation site for hen lysozyme folding.     

Analyses of Native Disulfide Bond Formations in the Early Stage of the Folding Process in Mutant Lysozymes Where the Long-Range Interactions in the Denatured State Were Modulated

In order to clarify whether modulation of long-range interactions in the denatured state affect native disulfide bond (SS bond) formations of hen egg white lysozyme (HEL) containing a pair of cysteine residues, we examined the extent of SS bond formation among 12 variants containing a pair of cysteines. The loss of clusters 5 and 6 in the denatured state affected the formation of Cys30-Cys115 and Cys6-Cys127 respectively.     

Analyses of native disulfide bond formations in the early stage of the folding process in mutant lysozymes where the long-range interactions in the denatured state were modulated

In order to clarify whether modulation of long-range interactions in the denatured state affect native disulfide bond (SS bond) formations of hen egg white lysozyme (HEL) containing a pair of cysteine residues, we examined the extent of SS bond formation among 12 variants containing a pair of cysteines. The loss of clusters 5 and 6 in the denatured state affected the formation of Cys30-Cys115 and Cys6-Cys127 respectively.     

NMR structural study of two-disulfide variant of hen lysozyme: 2SS[6-127, 30-115]--a disulfide intermediate with a partly unfolded structure

The 15N-labeled recombinant hen lysozyme and two species of two-disulfide variants, denoted as 2SS[6-127, 30-115] and 2SS[64-80, 76-94], were studied by means of NMR spectroscopy. The former variant contains two disulfide bridges in the alpha-domain, while the latter has one disulfide bridge in the beta-domain and the other one at the interface between two domains. Resonance assignments were performed using 3D TOCSY-HSQC and NOESY-HSQC spectra. The 15N-1H-HSQC spectrum of 2SS[6-127, 30-115] was similar to that of recombinant lysozyme as a whole, although a number of cross-peaks disappeared. On the other hand, the HSQC spectrum of 2SS[64-80, 76-94] was characteristic of unfolded proteins. The structure of 2SS[6-127, 30-115] was thoroughly examined on the basis of NOE contacts determined by NMR spectroscopy. The structure of the alpha-domain was quite similar to that of authentic lysozyme, while the beta-domain was largely unstructured. However, NMR data clearly demonstrated that some residual structures exist in the beta-domain. The beta1 and beta2 strands were maintained stably as an antiparallel beta-sheet. In addition, the residues 55 and 56 were located in the vicinity of the end of the B-helix. Further, the C-helix was properly set with side-chains of I88, V92, K96, and V99 facing toward the hydrophobic core in the alpha-domain. These residual structures inherent in the amino acid sequence were evaluated concerning the folding process of lysozyme. Our experiments imply that the establishment of the backbone conformation ranging from residues 76-99 plays a key role in attaining the cooperativity between two domains required for the folding transition.     

Two-State Folding of Lysozyme Versus Multiple-State Folding of α-Lactalbumin Illustrated by the Technique of Disulfide Scrambling

The folding of lysozyme and of alpha-lactalbumin exhibits vastly different kinetics and pathways. Existing evidence indicates that folding intermediates of alphaLA form a well-populated equilibrium molten globule state that is absent in the case of hen lysozyme. We demonstrate here such divergent folding mechanisms of lysozyme and alphaLA using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of lysozyme (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127) and alphaLA (Cys6-Cys28, Cys61-Cys73, Cys77-Cys91, Cys111-Cys120) were allowed to refold in parallel to form the native protein. Folding kinetics was measured by the recovery of the native structure. Folding intermediates, which illustrate the folding pathway, were trapped by quenching disulfide shuffling and were analyzed by reversed-phase high-pressure liquid chromatography. The results revealed that under identical folding conditions, the folding rate of lysozyme is about 30-fold faster than that of alphaLA. Folding intermediates of lysozyme are far less heterogeneous and sparsely populated than those of alphaLA. Numerous predominant on-pathway and off-pathway intermediates observed along the folding pathway of alphaLA are conspicuously absent in the case of lysozyme. The difference is most striking under fast folding conditions performed in the presence of protein disulfide isomerase. Under these conditions, folding of lysozyme undergoes a near two-state mechanism without accumulation of stable folding intermediates.     

Native-like tertiary structure formation in the alpha-domain of a hen lysozyme two-disulfide variant.

Structure formation in two species of the two-disulfide variant of hen lysozyme was investigated by means of CD spectroscopy, disulfide exchange measurement, and 1H-NMR spectroscopy. One species, 2SS [6-127, 30-115], which contained the two disulfide bonds found in the alpha-domain of authentic lysozyme, had amounts of secondary and tertiary structures, and bacteriolytic activity comparable to those of authentic lysozyme, and showed a cooperative thermal unfolding. By contrast, the other species, 2SS [64-80, 76-94], which contained the beta-domain disulfide bond as well as the inter-domain one, had a limited amount of secondary structure and little tertiary structure. Disulfide-exchange did not occur for 2SS [6-127, 30-115], whereas it occurred for 2SS [64-80, 76-94], indicating that the protein main-chain fold coupled with the formation of two disulfide bonds is relatively stable for the former variant, while unstable for the latter. 1H-NMR spectra of 2SS [6-127, 30-115] showed that native-like local environment is present within the region that corresponds to the alpha-domain, while it is absent within the region that corresponds to the beta or inter-domain. These results indicate that the alpha-domain of hen lysozyme can be an independent folding domain at equilibrium. Although the bipartite nature in the structure formation of hen lysozyme is similar to that reported for alpha-lactalbumin, differences exist between the disulfide-intermediates of the two proteins in terms of the structural domain that accomplishes tertiary structure.     

Specific disulfide cleavage is required for ubiquitin conjugation and degradation of lysozyme.

Both ubiquitin conjugation and ubiquitin-dependent degradation of chicken egg white lysozyme in a reticulocyte lysate depend on the presence of a reducing agent. We present evidence that the reduction of a specific disulfide bond, namely that at Cys6-Cys127, facilitates ubiquitination and is a prerequisite to the formation of a multiubiquitin chain on one of at least four chain initiation sites on lysozyme. The Cys6-Cys127 disulfide bond in lysozyme can be specifically reduced, and the modified protein can be isolated after carboxymethylation of the 2 resulting cysteines. This modified lysozyme no longer requires the presence of a reducing agent for ubiquitin conjugation and degradation. Inhibition of ubiquitination by the dipeptide Lys-Ala revealed that this modified lysozyme, like the unmodified protein, is recognized via the binding of the ubiquitin protein ligase, E3, to the substrate's N-terminal lysyl residue. Both the rate and the extent of ubiquitin-lysozyme conjugation, however, are significantly higher with this modified substrate. Likewise, ubiquitin-dependent degradation of 6,127-reduced/carboxymethylated lysozyme was 2-4-fold faster than degradation of the unmodified counterpart. These results are consistent with an interpretation that the modified lysozyme mimics an intermediate formed at the rate-limiting step of the degradation of lysozyme in the reticulocyte lysate. Reduction of the Cys6-Cys127 disulfide bond is expected to unhinge the N-terminal region of lysozyme, and we propose that the recognition of this otherwise stable protein by the ubiquitin pathway is due to facilitated binding of E3 that results from such a conformational transition.     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Location of disulfide bonds in mature alpha-L-fucosidase from pea.

Fuc-9 is the mature form of a vacuolar alpha-L-fucosidase enzyme which seems to play an important role in plant growth regulation. Fuc-9 is a 202-residue protein containing five Cys residues located at positions 64, 109, 127, 162 and 169. In this study, the disulfide structure of Fuc-9 was determined by MALDI-TOF mass spectrometry (MS), with minimal clean-up of the samples and at a nanomolar scale. Two strategies, based on a specific chemical cleavage (with 2-nitro-5-thiocyanobenzoic acid and alkaline conditions) at the Cys residues and modification of Cys residues by acrylamide/deuterium labeled acrylamide alkylation, were used. Using these methods, the disulfide pairings Cys64-Cys109 and Cys162-Cys169 could be established. The advantages and limitations of our experimental approach are discussed.     

A study of renaturation of reduced hen egg white lysozyme. Enzymically active intermediates formed during oxidation of the reduced protein.

The material obtained from reduced hen egg white lysozyme after complete air oxidation at pH 8.0 and 37 degrees has yielded, by gel filtration on a Bio-Gel P-30 column, enzymically active species and an enzymically inactive form which eluted sooner than the active species but later than expected for a dimer of lysozyme. Reduced lysozyme also elutes at the same position as this inactive material. Examination of the fragments produced on CNBr cleavage of the inactive form indicates that at least 24% of the population contains incorrect disulfide bonds involving half-cystine residues 6, 30, 115, and 127. Tryptophan fluorescence and the intrinsic viscosity of the inactive form show an enlarged molecular domain with a disordered conformation. The yield of the inactive form increases as the oxidation of reduced lysozyme is accelerated using cupric ion. In the presence of 4 X 10(-5) M cupric ion, reduced lysozyme forms almost quantitatively the inactive form, which is almost completely converted to the native form by sulfhydryl-disulfide interchange catalyzed by thiol groups of either reduced lysozyme or beta-mercaptoethanol. The material trapped by alkylation of the free sulfhydryl groups with [1-14C]iodoacetic acid during the early stage of air oxidation of reduced lysozyme was fractionated by gel filtration to permit separation of the active species from the inactive form. Ion exchange chromatography of the active species yielded completely renatured lysozyme and three major enzymically active radioactive derivatives. Two of these derivatives contained approximately 2 mol of S-carboxymethylcysteine. Isolation and characterization of radioactive tryptic peptides from each of the three active forms, permitted the identification of Cys 6 and Cys 127, Cys 76 and 94, and Cys 80 as the sulfhydryl groups alkylated in these three incompletely oxidized, partially active forms. Thus, it appears that the interatomic interactions maintaining the compact three-dimensional structure of native lysozyme are operational even when one of these three native disulfide bonds between Cys 6 and Cys 127, Cys 76 and Cys 94, and Cys 64 and 80 is open.     

A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23-231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23-124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23-231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.     

Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34.

Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure-activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ?i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C-terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.     

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.     

Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme

Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.     

Oxidative folding of human lysozyme: effects of the loss of two disulfide bonds and the introduction of a calcium-binding site

Mutant human lysozymes (HLZ) lacking two disulfide bonds were constructed to study the importance of each disulfide bond on oxidative refolding. To avoid destabilization, a calcium-binding site was introduced. Five of the six species of two-disulfide mutants could be obtained with enzymatic activity. Based on the information obtained from refolding and unfolding experiments, the order of importance in oxidative refolding was found to be as follows: SS2(Cys30-Cys116) > SS1(Cys6-Cys128) SS3(Cys65-Cys81) > SS4(Cys77-Cys95). Without SS2, these mutants refolded with low efficiency or did not refold at all. The bond SS2 is located in the interface of B-and D-helices, and a small hydrophobic cluster is formed near SS2. This cluster may play an important role in the folding process and stabilization, and SS2 may act as a stabilizer through its polypeptide linkage. The bond SS2 is the most important disulfide bond for oxidative folding of lysozymes.     

Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep-CAM)

The GA733-2 antigen is a cell surface glycoprotein highly expressed on most human gastrointestinal carcinoma and at a lower level on most normal epithelia. It is an unusual cell-cell adhesion protein that does not exhibit any obvious relationship to the four known classes of adhesion molecules. In this study, the disulfide-bonding pattern of the GA733-2 antigen was determined using matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of purified tryptic peptides treated with 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially reduced and alkylated. Numbering GA733-2 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5, which is a novel pattern for a cysteine-rich domain instead of the expected epidermal growth factor-like disulfide structure. The next three disulfide linkages are Cys7-Cys8, Cys9-Cys10, and Cys11-Cys12, consistent with the recently determined disulfide pattern of the thyroglobulin type 1A domain of insulin-like growth factor-binding proteins 1 and 6. Analysis of glycosylation sites showed that GA733-2 antigen contained N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential N-linked glycosylation sites, Asn175 was not glycosylated, whereas Asn88 was completely glycosylated, and Asn51 was partially glycosylated. These data show that the extracellular domain of the GA733-2 antigen consists of three distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733 type 3 motif).     

Contribution of disulfide bridging to epitope expression of the dengue type 2 virus envelope glycoprotein

The individual contributions of each of the six conserved disulfide (SS) bonds in the dengue 2 virus envelope (E) glycoprotein (strain 16681) to epitope expression was determined by measuring the reactivities of a panel of well-defined monoclonal antibodies (MAbs) with LLC-MK(2) cells that had been transiently transformed with plasmid vectors expressing E proteins that were mutant in their SS bonds. Three domain I (DI) epitopes (C1, C3, and C4) were affected by elimination of any SS bond and were essentially the only epitopes affected by elimination of the amino-proximal SS1 formed between Cys 3 and Cys 30. The remaining DI epitope (C2) was sensitive to only SS3-bond (Cys 74-Cys 105) and SS6-bond (Cys 302-Cys 333) elimination. Of the four DII epitopes examined, reactivities of three anti-epitope MAbs (A1, A2, and A5) were reduced by elimination of SS2 (Cys 61-Cys 121), SS3, SS4 (Cys 94-Cys 116), SS5 (Cys 185-Cys 285), or SS6. The other DII epitope examined (A3) was sensitive only to SS2- and SS3-bond elimination. The three DIII epitopes tested (B2, B3, and B4) were most sensitive to elimination of SS6. The flavivirus group epitope (A1) was less sensitive to elimination of SS3 and SS6. This result may indicate that the region proximal to the E-protein fusion motif (amino acids 98 to 110) may have important linear components. If this observation can be confirmed, peptide mimics from this region of E protein might be able to interfere with flavivirus replication.     

Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: implications for formation of the molten globule state

Molten globule states are partially folded states of proteins which are compact and contain a high degree of secondary structure but which lack many of the fixed tertiary interactions associated with the native state. A set of peptides has been prepared in order to probe the role of local interactions in the vicinity of the Cys(6)-Cys(120) disulfide bond in stabilizing the molten globule state of human alpha-lactalbumin. Peptides derived from the N-terminal and C-terminal regions of human alpha-lactalbumin have been analyzed using nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy and sedimentation equilibrium experiments. A peptide corresponding to the first helical region in the native protein, residues 1-13, is only slightly helical in isolation. Extending the peptide to include residues 14-18 results in a modest increase in helicity. A peptide derived from the C-terminal 12 residues, residues 112-123, is predominantly unstructured. Crosslinking the N- and C-terminal peptides by the native disulfide bond results in almost no increase in structure and there is no evidence for any significant cooperative structure formation over the range of pH 2.2-11.7. These results demonstrate that there is very little enhancement of local structure due to the formation of the Cys(6)-Cys(120) disulfide bond. This is in striking contrast to peptides derived from the region of the Cys(28)-Cys(111) disulfide.     

Convergent solid-phase synthesis of hirudin.

Hirudin variant 1 (HV1), a small protein consisting of 65 amino acids and three disulfide bonds, was synthesized by using Fmoc-based convergent methods on 2-chlorotrityl resin (CLTR). The linear sequence was assembled by the sequential condensation of 7 protected fragments, on the resin-bound 55-65 fragment. The conditions of fragment assembly were carefully studied to determine the most efficient synthetic protocol. Crude reduced [Cys(16, 28)(Acm)]-HV1 thus obtained was easily purified to homogeneity by RP-HPLC. Disulfide bridges were successfully formed by a two-step procedure, involving an oxidative folding step to form Cys(6)-Cys(14) and Cys(22)-Cys(39) linkages, followed by iodine oxidation to form the Cys(16)-Cys(28) bond. The correct disulfide bond alignment was established by peptide mapping using Staphylococcus aureus V8 protease at pH 4.5.